THE NUCLEUS & NUCLEOCYTOPLASMIC TRANSPORT Nuclear Trans lec 23... · THE NUCLEUS &...

THE NUCLEUS & NUCLEOCYTOPLASMIC TRANSPORT

1. Introductiona. The nucleusb. The nuclear envelopec. Transport cargoes

2. The solid phase: the nuclear pore complex (NPC)

3. Signalsa. Nuclear localization signals (NLSs)b. Nuclear export signals (NESs)

4. The soluble phase: nuclear transport factorsa. Importins and exportinsb. The GTPase Ran

5. Mechanism of transport through the NPC

6. Segregation of nuclear components during cell division

a. The Nuclear Envelope1. Introduction

mRNAs

snRNAssnRNPs

tRNAssnRNPs

snRNAs

Proteins

Proteins

IonsSmall molecules

(<60kD)

Karyophilic proteins(NLS)

RibosomalProteins

RibosomalSubunits

rRNAs

5SrRNAs

Nuc leolus

b. Transport Cargoes

2. The solid phase: the nuclear pore complex (NPC)

Cytoplasmic side

Nuclear side

The nuclear pore has a 9nm wide aqueous diffusion channel

3. Signals: how were they identified?

IMPORTIN

EXPORTIN

IMPORT EXPORT

NUCLEUS

CYTOPLASM

NUCLEUS

CYTOPLASM

EXPORTIN

IMPORTINIMPORTCARGO

IMPORTCARGO

EXPORTCARGO

EXPORTCARGO

4. The soluble phase a. Importins and Exportins

Ran-GTPRan-GTPH2O

Pi GTP

GDPRanGAP 1

RanGEF:

RCC1

CYTOPLASMRan-GDPRan-GDP

NUCLEUS

4. The soluble phase b. The GTPase Ran

IMPORTIN

EXPORTIN

IMPORT EXPORT

RanGTP-binding and import cargo release

RanGTP-hydrolysis and export cargo release

NUCLEUS

CYTOPLASM

NUCLEUS

CYTOPLASM

EXPORTIN

GTPGTP

GDP

GDP

IMPORTINIMPORTCARGO

IMPORTCARGO

EXPORTCARGO

EXPORTCARGO

Ran regulates substrate binding and release4. The soluble phase

5. A model for translocation through the NPC

Example: Reversible nuclear accumulation of NF-AT

Compartmentalisation allows the regulation of gene expression

SUMMARY

I. Signals target proteins into and out of the nucleusa. active transportb. signals are necessary and sufficient

II. Transport occurs through the nuclear pore complex (NPC), a large multi-protein complexa. NPC has aqueous channelb. no unfolding of cargoes is requiredc. transport is bi-directional

III. Signals are recognized by soluble receptors:Importins and exportins

a. bind specific classes of cargob. shuttle between cytoplasm and nucleusc. interact with nucleoporins

IV. Ran-GTP defines the nucleoplasm and the perichromatin spacea. Ran-GTP is asymmetrically distributedb. Ran regulates cargo binding and release

Visualization of Transcription in Cells or Tissue Sections

•3H-Uridine Incorporation•Br-UTP Incorporation•Fluorescence In Situ Hybridization

Visualizing Gene Expression in Living Cells

Janicki et al. (2004) Cell 116, 683-698

2.5 hrs. post-transfection (-) Dox

CFP-LacRepressor

MS2 BindingProtein-YFP

Merge

Janicki et al. (2004) Cell 116, 683-698.

CFP-Lac RepressorCFP-SKL

MS2 BindingProtein-YFP

Merge

2.5 hrs. post-transfection + 2.5 hrs. after theaddition of Dox

Janicki et al. (2004) Cell 116, 683-698.

CFP-lac repressorCFP-SKL

YFP-RNA pol II Merge

The RNA polymerase II large subunit isrecruited to the active locus

Janicki et al. (2004) Cell 116, 683-698.

Fluorescence Recovery after photobleaching (FRAP)

FRAP analysis of a pre-mRNA splicing factorin the cell nucleus

SF2/ASF has a half-time of fluorescence recovery of approximately1.8 (+/- 0.6) seconds.

Bubulya et al. (2004) J. Cell Biol. 167, 51-63.

SUMMARY

I. Single Cell Imaging of Gene Regulation

II. Development of in vivo fluorescent tags

III. New ways to amplify gene loci and detecttranscripts

IV. Clever ways of using RNA binding proteins to localize RNA products

V. Using laser to bleach and measure recovery of proteins to a locus

VI. Fluorescence in situ hybridization (FISH) and multi-copy gene arrays to localize specific genes